The Inheritance and Expression of cry1A Gene in Transgenic Maize

We investigated the inheritance and expression of cry1A gene in transgenic maize ( Zea mays L.) by Southern blotting analysis and enzyme_linked immunosorbent assays (ELISA). The results showed that cry1A had been transmitted to progeny of transgenic maize as a single gene. Contents of cry1A insecticidal protein were significantly different among transgenic maize lines and various tissues of the same transgenic lines. High expression of cry1A protein occurred in green tissues, such as leaf and husk leaf, and low expression occurred in pith, tassel, ear pith, pollen and silk. The results also showed that the contents of cry1A insecticidal protein in leaves of transgenic maize increased with the advance of development and there was no significant difference in cry1A expression level among various generations of transgenic maize.

The Influence of Bt-Transgenic Maize Pollen on the Bacterial Diversity in the Midgut of Chinese Honeybees, Apis cerana cerana

Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear crylAh gene （800 ng mL^-1） and supercoiled plasmid DNA （800 ng mL^-1） under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon＇s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic crylAh maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.

Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS） encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops（Cao et al. 2012）. In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301 Ubi Sp AM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase（rbcS） small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize（Zea mays） immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

Effect of three insect-resistant maizes expressing Cry1le,Cry1Ab/Cry2Aj and Cry1Ab on the growth and development of armyworm Mythimna separata(Walker)

Three transgenic maize events(IE09S034,Shuangkang 12--5 and C0030.3.5)produced Cry1le,Cry1Ab/Cry2Aj and G10-EPSPS,Cry1Ab and EPSPS,respectively,all of which target the Asian corn borer.The oriental armyworm Mythimna separata(Walker)is the secondary target.In this study,the effects of the three Bt maizes on the development and survival of armyworm were studied.The results showed that IE09S034 had insecticidal activity against 1st instar larvae,and the survival rate of armyworm fed with Bt maize for 10 days was 462%,significantly lower than that of the control.The larvae at 3rd--6th instar were more tolerant of the Bt toxin than the early instar larvae.However,Shuangkang 12-5 had good insecticidal activity against 1st-5th instar larvae.The mortality was nearly 100%when the larvae were fed with Shuangkang 12-5 before 3rd instar,and the toxin had quick acting eficacy.This event significantly ihibited the development of armyworm;that is,the larval duration of the 3rd and 4th instar larvae fed with Shuangkang 12-5 was prolonged by 4.5 and 3.0 days,respectively.The pupal weight and egg number were also significantly lower than those of the control.For C0030.3.5,it could control 1st--5th instar larvae effectively.The mortality rates were all over 50%if 1st-3rd larvae were fed with this event.The pupal weight of 4th-6th instar larvae fed with Bt maize were only 53.9,56.8 and 54.6%,respectively,compared to that of the control.The number of eggs laid was significantly less than the control.The results indicate that all three transgenic maize events exhibit the potential to provide effective control of early instar larvae of armyworm.which can be commercialized in future to control lepidoptera pests such as Asian corn borer and armyworm.

Screening Method for Transgenic Maize with Kanamycin Resistance

[Objective]The paper was to establish simple and effective method to screen marker gene in maize with kanamycin resistance.[Method]Using inbred line "Chang 7-2" and hybrid "Zhengdan 958" of maize as test materials,their seeds were soaked with different concentrations and volumes of kanamycin for3 and 4 d,respectively,the rate of albino seedlings and average seedling height after sowing for10 d were investigated.[Result]The rate of albino seedlings not only was related to kanamycin concentration,but also had relationship with solution volume during soaking process.The difference between inbred line and hybrid was no significant.When 100 ml of kanamycin solution with concentration of 200 mg/L was used to soak seeds for 3 d,the rate of albino seedlings basically could reach 100%.When 100 ml of kanamycin solution with concentration of 100 mg/L was used to soak 20 seeds for 3 d to carry out resistance screening,the accuracy was up to 84.8% after verifying the screening test of T1 transgenic maize plants.[Conclusion]The method was feasible,which could be used as a simple method for screening transgenic gene maize with kanamycin resistance.

Construction of Maize Universal Expression Vector PGM-35Sbar and Maize Transformation

[Objective] The paper was to construct maize universal expression vector, in order to provide basis for using transgenic methods to improve abiotic stress tolerance of maize. [Method] Based on the transformation of existing pGreen0229 plant expression vector, phosphinothricin-resistant selectable marker-bar gene driving by CaMv35S promoter was constructed, which could be used to connect target gene of maize expression vector PGM-35Sbar, and transform Ji444 maize inbred lines by pollen tube pathway. [Result] The universal expression vector for PGM-35Sbar maize had been successfully constructed. When the maize plants were transformed, 14 resistant plants were obtained, and 12 plants were identified to be positive plants by PCR detection. [Conclusion] The study provided basis for rapid construction of maize expression vector containing specific target gene.

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses

Aldehyde dehydrogenases（ALDHs） represent a large protein family, which includes several members that catalyze the oxidation of an aldehyde to its corresponding carboxylic acid in plants. Genes encoding members of the ALDH7 subfamily have been suggested to play important roles in various stress adaptations in plants. In this study, quantitative RT-PCR analysis revealed that a maize ALDH7 subfamily member（ZmALDH7B6） was constitutively expressed in various organs, including roots, leaves, immature ears, tassels, and developing seeds. The abundance of ZmALDH7B6 mRNA transcripts in maize roots was increased by ammonium, NaCl, and mannitol treatments. To further analyze tissue-specific and stress-induced expression patterns, the 1.5-kb 5′-flanking ZmALDH7B6 promoter region was fused to the β-glucuronidase（GUS） reporter gene and introduced into maize plants. In roots of independent transgenic lines, there was significant induction of GUS activity in response to ammonium supply, confirming ammonium-dependent expression of ZmALDH7B6 at the transcript level. Histochemical staining showed that GUS activity driven by the ZmALDH7B6 promoter was mainly localized in the vascular tissues of maize roots. These results suggested that ZmALDH7B6 is induced by multiple environmental stresses in maize roots, and may play a role in detoxifying aldehydes, particularly in vascular tissue.

Quantification of CrylAb Toxin in Bt Maize for Ostrinia nubilalis （Lep.： Crambidae） Bioassay

Two variants of diet composition were prepared to evaluate the susceptibility of ECBs to CrylAb toxin as follows： 1） 38-0600 Stonefly Heliothis Diet mixed with purified CrylAb protein and 2） 38-0600 Stonefly Heliothis Diet mixed with lyophylized leaves of Bt maize MON 810-YieldGard. A method of sample preparation and extraction of Bt toxin for reproducible ELISA quantification were optimized. The qualitative DAS-ELISA kit from Agdia was optimized for use in quantitative analysis of Cry lAb toxin. The mortality of ECB larvae from the laboratory strain on the diet with CrylAb toxin did not differ significantly from the mortality on the diet with Bt maize leaves with the same rate of Cry lAb toxin. Similarly, the mortality of the ECB larvae from the field population on the diet with Bt maize leaves did not differ significantly from the mortality of ECB larvae from the laboratory strain on the same type of diet. Therefore, the incorporation of Bt maize leaves into the diet did not influence the efficacy of CrylAb toxin against ECBs. Using this method, a susceptibility of one field population of ECBs from the Czech Republic to CrylAb toxin was determined （LC50 of 2.16 μg of Cry 1Ab g^-1 of diet）.

Uncertainty in Detecting Specific Fragment of Genetically Modified Maize Event NK603 by Real-time Fluorescence Quantitative PCR

In this study, the event-specific real-time fluorescence quantitative PCR method established by Siehuan Academy of Agricultural Sciences was employed to detect the content of flanking fragment （specific fragment） in samples containing 2% genetically modified maize event NK603. The uncertainty of detection results was evaluated based on various uncertainty sources, such as PCR amplification system, data analysis and micropipette. The results showed that A-type uncertainty （ uA ）, B-type uncertainty （ uB ）, combined standard uncertainty （ uC ） and expanded uncertainty （ U95 ） were 0. 000 8,0.1301,0. 001 and 0. 002, respectively; the final detection result was 1.9% ±0.002. Thus, the main uncertainty in detecting flanking fragment of genetically modified maize event NK603 with realtime fluorescence quantitative PCR method was derived from the random effect in the experimental process.

Recombinant protein detection and its content in total protein, lipids and toxic antinutritional substances in Mexican maize

The engineering genetic technology has developed Bt maize events which contain recombinant protein that will be safe for the consumer. The aflatoxins are contaminants present in maize capable of producing cancer and decreasing the immune response in human, additionally contained polyphenols compounds considered non nutritive. The objective of this study was to identify the presence of recombinant protein in hybrid and local varieties of corn and evaluate the content of aflatoxins and tannins. 25 samples of white grain maize for human consumption were collected, 12 were for hybrid maize and 13 local varieties, from the states of Hidalgo, Mexico and Morelos. Samples were analyzed for Cry1Ab/Cry1Ac, using lateral flow strip method, crude protein and lipids by standard methods. Aflatoxins were assessed by comercial Elisa kit and tannins by spectroscopy method. The data were grouped in a completely random model and an analysis of variance was performed. The results indicated that 44.5% of hybrid corn was positive by Bt-Cry1Ab/1Ac proteins, containing 9.02% ± 2.5 lipids and 11.33% ± 2.2 crude protein, 189 ± 0.92 mg/g of tannins and 6.36 ± 3.3 μg·g-1 aflatoxins. The local maize samples (55.5%) were negative to Bt-Cry1Ab/ Cry1Ac, which protein content was of 8.68% ± 0.90, 6.14% lipids ± 2.3, 273 ± 0.40 mg/100g tannin and 7.15 ± 3.3 μg·g-1 of aflatoxins. In conclusion, we observed an improvement of nutrient composition in hybrid maize with Bt proteins, and decrease in tannins content comparing with some local varieties without Bt proteins. The effectiveness of Bt maize expressing the Cry1Ab/ Cry1Ac in reducing aflatoxin contamination was not observed, therefore, additive affects of aflatoxins contamination in maize Bt-Cry need to be further investigated in cancer disease development.

Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein CrylAb/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer （Ostnnia furnacalis）, cotton boll worm （Helicoverpa armigera）, and armyworm （Mythimna separata）. This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.

In maize,co-expression of GAT and GR79-EPSPS provides high glyphosate resistance,along with low glyphosate residues

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified(GM)crops.Herbicide-tolerant crops,especially glyphosate-resistant crops,offer great advantages for weed management;however,despite these benefits,glyphosate-resistant maize(Zea mays L.)has not yet been commercially deployed in China.To develop a new bio-breeding resource for glyphosate-resistant maize,we introduced a codon-optimized glyphosate N-acetyltransferase gene,gat,and the enolpyruvyl-shikimate-3-phosphate synthase gene,gr79-epsps,into the maize variety B104.We selected a genetically stable high glyphosate resistance(GR)transgenic event,designated GG2,from the transgenic maize population through screening with high doses of glyphosate.A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome,and these two genes were stably transcribed and translated.Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent(a.e.)glyphosate per ha with no effect on phenotype or yield.A gas chromatography-mass spectrometry(GC–MS)analysis revealed that,shortly after glyphosate application,the glyphosate(PMG)and aminomethylphosphonic acid(AMPA)residues in GG2 leaves decreased by more than 90%compared to their levels in HGK60 transgenic plants,which only harbored the epsps gene.Additionally,PMG and its metabolic residues(AMPA and N-acetyl-PMG)were not detected in the silage or seeds of GG2,even when far more than the recommended agricultural dose of glyphosate was applied.The co-expression of gat and gr79-epsps,therefore,confers GG2 with high GR and a low risk of herbicide residue accumulation,making this germplasm a valuable GR event in herbicide-tolerant maize breeding.

Field observations of Ostrinia nubilalis eclosion and post-eclosion activity of females around their natal plants

The early part of the post-eclosion, pre-mating period were examined under field conditions for Ostrinia nubilalis. Post-eclosion behavior of 25 and 21 females during the first and second flight periods were observed until they left their natal site. Summer generation larvae were reared under field conditions and the timing of adult eclosion was observed. Eclosion occurred at two times during the day, peaking before dawn and before dusk; 46% of females and 56% of males eclosed during the morning period and the rest eclosed during the evening period. After eclosion, females spent 30-60 min expanding their wings. Their typical behavior was to remain calmly on their natal site. None of the females exhibited calling behavior before leaving. All females left their natal sites sometime before dawn. The probability of leaving increased with time. Leaving rates were not significantly different between females of the first and second flight. These field observations indicate that females have several possibilities for pre-mating movement, which might allow females to move out from their natal field before mating. In addition, we also discuss the influence of pre-mating movements in relation to the rate of Bacillus thuringiensis （Bt） resistance evolution.